Recombinant expression of chlamydia momp antigen

ABSTRACT

The present invention relates to methods for the recombinant expression of chlamydia major outer membrane protein (MOMP) comprising transforming a population of  E. coli  host cells with an expression vector comprising a nucleic acid molecule that encodes chlamydia MOMP and encodes a leader sequence for targeting the MOMP to the outer membrane of the cell, wherein the nucleic acid molecule is operatively linked to a promoter. The method of the invention allows expression of MOMP in the outer membrane of the cell, which leads to protein folding that is more like native MOMP relative to a MOMP protein that is expressed intracellularly. Also provided by the invention are uses of the recombinant MOMP in pharmaceutical compositions and methods for the treatment and/or prophylaxis of chlamydia infection and/or the effects thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is continuation of U.S. application Ser. No.17/127,669, filed Dec. 18, 2020, which is a continuation application ofU.S. application Ser. No. 16/894,968, filed Jun. 8, 2020, which is adivisional application of U.S. application Ser. No. 15/527,789, filedMay 18, 2017, which is a § 371 National Phase Application ofInternational Application No. PCT/US15/060780, filed Nov. 16, 2015,which claims the benefit of U.S. provisional application No. 62/082,889,filed Nov. 21, 2014, the contents of which are hereby incorporated byreference in their entirety.

FIELD OF THE INVENTION

The present invention relates to methods for the recombinant expressionof Chlamydia antigen MOMP and translocation to the outer membrane of E.coli, pharmaceutical compositions comprising recombinant MOMP and usesof the recombinant MOMP and pharmaceutical compositions of the inventionin methods for the prevention of Chlamydia infection and/or the clinicalmanifestations thereof.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The sequence listing of the present application is submittedelectronically via EFS-Web as an ASCII formatted sequence listing with afile name “23870USDIV-SEQLIST.TXT”, creation date of Jun. 4, 2020, and asize of 53 kB. This sequence listing submitted via EFS-Web is part ofthe specification and is herein incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

Chlamydia trachomatis is an obligate intracellular Gram-negativebacterium responsible for a number of pathologies, including oculartrachoma and several sexually transmitted diseases. There are manydifferent strains of C. trachomatis, which are separated into multipleserovars based on serological differences in the chlamydial major outermembrane protein (MOMP). C. trachomatis serovars A, B, Ba, and C areresponsible for ocular trachoma which can cause conjunctivitis,conjunctival scarring and corneal scarring. C. trachomatis serovars D,Da, E, F, G, H, I, Ia, J, Ja and K are responsible for oculogenitaldisease which can cause cervicitis, urethritis, endometritis, pelvicinflammatory disease, tubal infertility, ectopic pregnancy, neonatalconjunctivitis and infant pneumonia. Chlamydia trachomatis serovars L1,L2 and L3 are responsible for lymphogranuloma venereum, which can causesubmocosa and lymph-node invasion, with necrotizing granulomas andfibrosis. (Reviewed in Brunham et al., Nature Reviews Immunology5:149-161, 2005; Montoya, Chlamydia, p. 694-702, In Wilson et al., Eds.Current Diagnosis & Treatment in Infectious Diseases, The McGraw-HillCompanies, Inc. 2001.) Asymptomatic genital Chlamydia infections arealso common, which may lead to infertility in women that are leftuntreated.

Chlamydia trachomatis infects mucosal epithelial cells. Like otherChlamydia, C. trachomatis undergoes a biphasic development cycle inwhich it begins the cycle as a metabolically inactive infectiouselementary body (EB) and transforms into a metabolically activereticulate body (RB). The bacterium exists outside the host cell as anEB, which is internalized by a host cell and surrounded by an endosomalmembrane forming an inclusion body, where the EB transforms into ametabolically active RB. The RB can divide by binary fusion. Withinabout 40-48 hours, the RB transforms back to an EB, which is released bythe host cell and can infect neighboring cells. (Id.)

Chlamydia MOMPs are part of a larger family of genetically related outermembrane proteins (the OmpA family) that are heat-modifiable, surfaceexposed porin proteins. OmpA proteins have a structurally similarN-terminal domain that is embedded in the bacterial outer membrane. OmpAproteins have been targeted for vaccine development because of theirsurface exposure, high immunogenicity, and role in the interactionbetween the bacteria and their host cells. Specifically, Chlamydia MOMPhas been a vaccine target for many researchers (Cambridge et al., Int.J. Nanomedicine 8:1759-71 (2013); Farris et al., Infection and Immunity79(3): 986-996 (2011); Hickey et al., Vaccine 22:4306-4315 (2004);Kalbina et al., Protein Expression and Purification 80: 194-202 (2011);O'Meara et al., PLOS One 8(4): 1-14; Skelding et al., Vaccine 24:355-366(2006), Tifrea et al., Infection and Immunity 81(5): 1741-1750 (2013)).However, a safe and effective Chlamydia vaccine remains unavailable toreduce the risk of Chlamydia infection or its associated pathogeniceffects. Additional vaccine candidates and methods for making them aretherefore needed.

SUMMARY OF THE INVENTION

The present invention is related to a method for the recombinantexpression of Chlamydia major outer membrane protein (MOMP) comprising:(a) transforming a population of E. coli host cells with an expressionvector comprising a nucleic acid molecule comprising a sequence ofnucleotides that encode a leader sequence for targeting the MOMP to theouter membrane of the cell and a sequence of nucleotides that encodeChlamydia MOMP, wherein the nucleic acid molecule is operatively linkedto a promoter; (b) culturing the transformed cells under conditions thatpermit expression of the nucleic acid molecule and translocation to theouter membrane of the cells to produce a recombinant Chlamydia MOMP; and(c) optionally purifying the MOMP. The method of the invention allowsrecombinant expression of MOMP in the outer membrane of the cell, whichleads to protein folding that is more like native MOMP relative to arecombinant MOMP protein that is expressed intracellularly.

In some embodiments of the invention, the nucleotide sequence encodingMOMP and/or the nucleotide sequence encoding the leader sequence iscodon harmonized. In alternative embodiments, the nucleotide sequenceencoding MOMP and/or the nucleotide sequence encoding the leadersequence is codon optimized.

In some embodiments of the invention, the leader sequence comprises theShigella flexneri SopA sequence, the Salmonella enterica PgtE sequence,the Yersinia pestis Pla, the E. coli OmpP sequence, the E. coli OmpAsequence, or the pectate lysase B (PelB) sequence. In furtherembodiments, expression of the recombinant MOMP is optimized by using alow or moderate strength promoter. In additional embodiments, optimizedexpression is achieved through using a vector that is characterized by atranscription/translation rate that is constrainable to low or moderate.

Also provided herein are recombinant MOMPs produced by the methods ofthe invention and pharmaceutical compositions comprising an effectiveamount of a recombinant MOMP of the invention and a pharmaceuticallyacceptable carrier.

In a further aspect, the invention provides methods for the treatment orprophylaxis of Chlamydia in a patient by administering a recombinantMOMP or a pharmaceutical composition of the invention to the patient.

As used throughout the specification and in the appended claims, thesingular forms “a,” “an,” and “the” include the plural reference unlessthe context clearly dictates otherwise.

As used throughout the specification and appended claims, the followingdefinitions and abbreviations apply:

As used herein, the term “recombinant” refers to a polypeptide ornucleic acid that does not exist in nature. The term “recombinant”polypeptide refers to a polypeptide that is prepared, expressed,created, or isolated by recombinant means, such as polypeptidesexpressed using a recombinant expression vector transfected into a hostcell. A recombinant polynucleotide mal also include two or morenucleotide sequences artificially combined and present together in alonger polynucleotide sequence, wherein the two sequences are not foundtogether (e.g. attached or fused) in nature, e.g. a promoter and aheterologous nucleotide sequence encoding a polypeptide that arenormally not found together in nature or a vector and a heterologousnucleotide sequence.

As used herein, the terms “isolated” or “purified” refer to a molecule(e.g., nucleic acid, polypeptide, bacterial strain, etc.) that is atleast partially separated from other molecules normally associated withit in its native state. An “isolated or purified polypeptide” issubstantially free of other biological molecules naturally associatedwith the polypeptide such as nucleic acids, proteins, lipids,carbohydrates, cellular debris and growth media. An “isolated orpurified nucleic acid” is at least partially separated from nucleicacids which normally flank the polynucleotide in its native state. Thus,polynucleotides fused to regulatory or coding sequences with which theyare not normally associated, for example as the result of recombinanttechniques, are considered isolated herein. Such molecules areconsidered isolated even when present, for example in the chromosome ofa host cell, or in a nucleic acid solution. Generally, the terms“isolated” and “purified” are not intended to refer to a completeabsence of such material or to an absence of water, buffers, or salts,unless they are present in amounts that substantially interfere withexperimental or therapeutic use of the molecule.

As used herein, “homology” refers to sequence similarity between twopolynucleotide sequences or between two polypeptide sequences when theyare optimally aligned. When a position in both of the two comparedsequences is occupied by the same base or amino acid monomer subunit,e.g., if a position in each of two DNA molecules is occupied by adenine,then the molecules are homologous at that position. The percent ofhomology is the number of homologous positions shared by the twosequences divided by the total number of positions compared ×100. Forexample, if 6 of 10 of the positions in two sequences are matched orhomologous when the sequences are optimally aligned then the twosequences are 60% homologous. Generally, the comparison is made when twosequences are aligned to give maximum percent homology. Gaps (in eitherof the two sequences being matched) are allowed in maximizing matching;gap lengths of 5 or less are preferred with 2 or less being morepreferred. Alternatively and preferably, two protein sequences (orpolypeptide sequences derived from them of at least 30 amino acids inlength) are homologous, as this term is used herein, if they have analignment score of at more than 5 (in standard deviation units) usingthe program ALIGN with the mutation data matrix and a gap penalty of 6or greater. See Dayhoff, M. O., in Atlas of Protein Sequence andStructure, pp. 101-110 (Volume 5, National Biomedical ResearchFoundation (1972)) and Supplement 2 to this volume, pp. 1-10.

Sequence identity refers to the degree to which the amino acids of twopolypeptides are the same at equivalent positions when the two sequencesare optimally aligned. Sequence identity can be determined using a BLASTalgorithm wherein the parameters of the algorithm are selected to givethe largest match between the respective sequences over the entirelength of the respective reference sequences. The following referencesrelate to BLAST algorithms that are often used for sequence analysis:BLAST ALGORITHMS: Altschul, S. F., et al., (1990) J. Mol. Biol.215:403-410; Gish, W., et al., (1993) Nature Genet. 3:266-272; Madden,T. L., et al., (1996) Meth. Enzymol. 266:131-141; Altschul, S. F., etal., (1997) Nucleic Acids Res. 25:3389-3402; Zhang, J., et al., (1997)Genome Res. 7:649-656; Wootton, J. C., et al., (1993) Comput. Chem.17:149-163; Hancock, J. M. et al., (1994) Comput. Appl. Biosci.10:67-70; ALIGNMENT SCORING SYSTEMS: Dayhoff, M. O., et al., “A model ofevolutionary change in proteins.” in Atlas of Protein Sequence andStructure, (1978) vol. 5, suppl. 3. M. O. Dayhoff (ed.), pp. 345-352,Natl. Biomed. Res. Found., Washington, DC; Schwartz, R. M., et al.,“Matrices for detecting distant relationships.” in Atlas of ProteinSequence and Structure, (1978) vol. 5, suppl. 3.” M. O. Dayhoff (ed.),pp. 353-358, Natl. Biomed. Res. Found., Washington, DC; Altschul, S. F.,(1991) J. Mol. Biol. 219:555-565; States, D. J., et al., (1991) Methods3:66-70; Henikoff, S., et al., (1992) Proc. Natl. Acad. Sci. USA89:10915-10919; Altschul, S. F., et al., (1993) J. Mol. Evol.36:290-300; ALIGNMENT STATISTICS: Karlin, S., et al., (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268; Karlin, S., et al., (1993) Proc. Natl.Acad. Sci. USA 90:5873-5877; Dembo, A., et al., (1994) Ann. Prob.22:2022-2039; and Altschul, S. F. “Evaluating the statisticalsignificance of multiple distinct local alignments.” in Theoretical andComputational Methods in Genome Research (S. Suhai, ed.), (1997) pp.1-14, Plenum, New York.

The term “cassette” refers to a nucleic acid molecule which comprises atleast one nucleic acid sequence that is to be expressed, along with itstranscription and translational control sequences. Changing thecassette, will cause the vector into which is incorporated to direct theexpression of different sequence or combination of sequences. In thecontext of the present invention, the nucleic acid sequences present inthe cassette will usually encode any polypeptide of interest such as animmunogen. Because of the restriction sites engineered to be present atthe 5′ and 3′ ends, the cassette can be easily inserted, removed orreplaced with another cassette.

The term “promoter” refers to a recognition site on a DNA strand towhich an RNA polymerase binds. The promoter forms an initiation complexwith RNA polymerase to initiate and drive transcriptional activity. Thecomplex can be modified by activating sequences such as enhancers, orinhibiting sequences such as silencers.

“MAA” means an amorphous aluminum hydroxyphosphate sulfate adjuvant.

As used herein, an “ISCOM-type adjuvant” is an adjuvant comprising animmune stimulating complex (ISCOM), which is comprised of a saponin,cholesterol, and a phospholipid, which together form a characteristiccaged-like particle, having a unique spherical, caged-like structurethat contributes to its function (for review, see Barr and Mitchell,Immunology and Cell Biology 74: 8-25 (1996)). This term includes bothISCOM adjuvants, which are produced with an antigen and comprise antigenwithin the ISCOM particle and ISCOM matrix adjuvants, which are hollowISCOM-type adjuvants that are produced without antigen.

As used herein, the term “derivative” refers to a polypeptide having oneor more alterations, which can be changes in the amino acid sequence(including additions and deletions of amino acid residues) and/orchemical modifications, relative to a reference sequence (e.g., a leadersequence and/or MOMP sequence described herein). In preferredembodiments, the derivative is at least 85%, at least 90%, at least 95%,at least 97%, at least 99% identical to the original reference sequenceprior to alteration. In general, derivatives retain the activity of thereference sequence, e.g. inducing an immune response. As used herein,the term “derivative” is not limited to derivatives of a wild-type ornative reference sequence, but includes derivatives of a mutant sequenceas a reference sequence. In preferred embodiments, any specifiedmutations of the mutant reference sequence are maintained, butalterations/modifications relative to the mutant reference sequence areincluded in the derivative sequence at amino acid residues other thanthe specified mutations of the reference sequence. As used herein, theterm “derivative” also includes polynucleotides that have one or morealterations relative to a reference nucleotide sequence.

In one embodiment, a derivative is a polypeptide that has an amino acidsequence which differs from the base sequence from which it is derivedby one or more amino acid substitutions. Amino acid substitutions may be“conservative” (i.e. the amino is replaced with a different amino acidfrom the same class of amino acids (e.g., non-polar, polar/neutral,acidic and basic), an amino acid with broadly similar properties, orwith similar structure (aliphatic, hydroxyl or sulfur-containing,cyclic, aromatic, basic, and acidic)) or “non-conservative” (i.e. theamino acid is replaced with an amino acid of a different type). Broadlyspeaking, fewer non-conservative substitutions will be possible withoutaltering the biological activity of the polypeptide. Some embodiments ofthe invention include derivatives that include substitution of no morethan 25 amino acid residues, 20 amino acid residues, 15 amino acidresidues, 12 amino acid residues, 11 amino acid residues, 10 amino acidresidues, 9 amino acid residues, 8 amino acid residues, 7 amino acidresidues, 6 amino acid residues, 5 amino acid residues, 4 amino acidresidues, 3 amino acid residues, 2 amino acid residues, or 1 amino acidresidue that is/are substituted relative to a reference sequence.

In another embodiment, a derivative is a polypeptide that has an aminoacid sequence which differs from the base sequence from which it isderived by having one or more amino acid deletions and/or additions inany combination. Deleted or added amino acids can be either contiguousor individual residues. In some embodiments, no more than 25 amino acidresidues, no more than 20 amino acid residues, no more than 15 aminoacid residues, no more than 12 amino acid residues, no more than 10amino acid residues, no more than 8 amino acid residues, no more than 7amino acid residues, no more than 6 amino acid residues, no more than 5amino acid residues, no more than 4 amino acid residues, no more than 3amino acid residues, no more than 2 amino acid residues, or no more than1 amino acid residue is/are deleted or added relative to a referencesequence.

In another embodiment, a derivative is a polypeptide that has an aminoacid sequence which differs from the base sequence from which it isderived by having one or more chemical modifications of the protein.Chemical modifications include, but are not limited to, modification offunctional groups (such as alkylation, hydroxylation, phosphorylation,thiolation, carboxylation and the like), incorporation of unnaturalamino acids and/or their derivatives during protein synthesis and theuse of crosslinkers and other methods which impose conformationalconstraints on the polypeptides.

As used herein, the term “conservative substitution” refers tosubstitutions of amino acids in a protein with other amino acids havingsimilar characteristics (e.g. charge, side-chain size,hydrophobicity/hydrophilicity, backbone conformation and rigidity,etc.), such that the changes can frequently be made without altering orsubstantially altering the biological activity of the protein. Those ofskill in this art recognize that, in general, single amino acidsubstitutions in non-essential regions of a polypeptide do notsubstantially alter biological activity (see, e.g., Watson et al. (1987)Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224(4th Ed.)). In addition, substitutions of structurally or functionallysimilar amino acids are less likely to disrupt biological activity.Exemplary conservative substitutions are set forth in Table 1.

TABLE 1 Exemplary Conservative Amino Acid Substitutions Original residueConservative substitution Ala (A) Gly; Ser Arg (R) Lys; His Asn (N) Gln;His Asp (D) Glu; Asn Cys (C) Ser; Ala Gln (Q) Asn Glu (E) Asp; Gln Gly(G) Ala His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K) Arg;His Met (M) Leu; Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala Ser (S) ThrThr (T) Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile; Leu

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that not all progeny willhave precisely identical DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

The term “treatment” refers to both therapeutic treatment andprophylactic or preventative measures. Individuals “in need of”treatment include those already with a Chlamydia infection, whether ornot manifesting any clinical symptoms, as well as those at risk of beinginfected with Chlamydia. Treatment of a patient with the pharmaceuticalcompositions of the invention includes one or more of the following:inducing/increasing an immune response against Chlamydia in the patient,inducing/increasing a virus neutralizing antibody response against oneor more Chlamydia viruses, preventing, ameliorating, abrogating, orreducing the likelihood of the clinical manifestations of Chlamydia inpatients who have been infected with Chlamydia, preventing or reducingthe likelihood of developing oculogenital disease, cervicitis,urethritis, endometritis, pelvic inflammatory disease, tubalinfertility, ectopic pregnancy, neonatal conjunctivitis, infantpneumonia and/or other disease or complication associated with Chlamydiainfection, reducing the severity or duration of the clinical symptoms ofChlamydia infection and/or other disease or complication associated withChlamydia, and preventing or reducing the likelihood of Chlamydiainfection.

The term “therapeutically effective amount” or “effective amount” meanssufficient pharmaceutical composition comprising recombinant MOMP isintroduced to a patient to produce a desired effect, including, but notlimited to: inducing/increasing an immune response against Chlamydia inthe patient, inducing/increasing a neutralizing antibody responseagainst Chlamydia in a patient, preventing or reducing the likelihood ofChlamydia infection, preventing or reducing the likelihood of Chlamydiarecurrent infection, preventing, ameliorating or abrogating the clinicalmanifestations of Chlamydia infection in patients who have been infectedwith Chlamydia, preventing one or more of ocular trachoma,conjunctivitis, conjunctival scarring, corneal scarring, oculogenitaldisease, cervicitis, urethritis, endometritis, pelvic inflammatorydisease, tubal infertility, ectopic pregnancy, neonatal conjunctivitis,infant pneumonia, and lymphogranuloma venereum; reducing the severity orduration of disease associated with Chlamydia. One skilled in the artrecognizes that this level may vary.

“An immunologically effective amount” refers to the amount of animmunogen that can induce an immune response against the heterologouspolypeptide when administered to a patient that can protect the patientfrom infection by the pathogen that expresses the heterologouspolypeptide (including primary, recurrent and/or super-infections)and/or ameliorate at least one pathology associated with infectionand/or reduce the severity/length of infection in the patient. Theamount is sufficient to significantly reduce the likelihood or severityof an infection. Animal models known in the art can be used to assessthe protective effect of administration of immunogen. For example,immune sera or immune T cells from individuals administered theimmunogen can be assayed for neutralizing capacity by antibodies orcytotoxic T cells or cytokine producing capacity by immune T cells. Theassays commonly used for such evaluations include but not limited toviral neutralization assay, anti-viral antigen ELISA, interferon-gammacytokine ELISA, interferon-gamma (IFN-γ) ELISPOT, intracellularmulti-cytokine staining (ICS), and ⁵¹Chromium release cytotoxicityassay. Animal challenge models can also be used to determine animmunologically effective amount of immunogen.

The term “immune response” refers to a cell-mediated (T-cell) immuneresponse and/or an antibody (B-cell) response.

The term “patient” refers to any human being that is to receive thepharmaceutical compositions described herein, including bothimmunocompetent and immunocompromised individuals. As defined herein, a“patient” includes those already infected with Chlamydia, either throughnatural infection or vaccination or those that may subsequently beexposed.

Additional abbreviations employed herein include the following: CI isconfidence interval; Cm is Chlamydia muridarium; CtD is Chlamydiatrachomatis Serovar D, CtE is Chlamydia trachomatis Serovar E, FACS isfluorescent activated cell sorting; GFI is geometric mean fluorescenceintensity; IPTG is isopropyl β-D-1-thiogalactopyranoside; LPS islipopolysaccharide MOMP is major outer membrane protein; rMOMP isrecombinant MOMP; rCmMOMP is recombinant Chlamydia muridarium MOMP; nOMVis a native outer membrane vesicle from E. coli that does not contain arecombinant MOMP gene and OM is outer membrane.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of codon usage harmonization on rMOMP surfaceexpression level. FACS geometric mean (geomean) fluorescence intensity(GFI) is shown for harmonized or optimized rCmMOMP genes at T=0 and T=4hours after IPTG induction. Geometric means of GFI from threeindependent experiments were plotted with 95% confidence interval.

FIGS. 2A-D provide an evaluation of expression conditions for rCtE MOMPwith a PelB leader sequence, which was constructed in the pAVE029vector. FIG. 2A shows the surface expression (FACS GFI) of rCtE MOMPafter different IPTG induction times (T=0 and T=4 hours) when expressedat different temperatures (37° C.=circles, 30° C.=squares, 25°C.=triangles, 16° C.=downward pointing triangles). FIG. 2B shows thecell concentration (OD590, geomean, 95% CI) at different temperatures(37° C.=circles, 30° C.=squares) after T=0 and T=4 IPTG induction timeand indicates the cell fragility. FIG. 2C shows the surface expressionwith different cell densities at induction (T=0). FIG. 2D shows thesurface expression (FACS GFI, geomean, 95% CI) with different cellculture media.

FIGS. 3A and 3B show expression of rCtE-MOMP on E. coli outer membraneunder optimized expression conditions. An E. coli transformantexpressing rCtE-MOMP with a PelB leader constructed in a pAVE029 vectorwas grown in Cinnabar medium at 37° C. and induced by 1 mM IPTG at 30°C. for 4 hours when OD590 reaches ˜0.5. FIG. 3A shows a whole cell flowcytometry binding histogram using anti-CtE EB mouse sera and a negativecontrol antibody at the end of induction. The geomean fluorescenceintensity is provided in FIG. 3B.

FIGS. 4A-D show expression of purified rCtE-MOMP. (FIG. 4A) SDS-PAGE;(FIG. 4B) anti-CtE EB mouse sera western blot; (FIG. 4C) anti-E. colicontrol nOMV (native outer membrane vesicle from E. coli that does notcontain recombinant MOMP gene) mouse sera; (D) anti-E. coli LPSmonoclonal antibody. Sample 1, control nMOMP; sample 2, purified rMOMP;sample 3, E. coli whole cell lysate; all samples are heated and reduced.Monomeric and dimeric forms of MOMP are indicated.

FIGS. 5A-C shows serum antibody responses against CtD EB in immunizedmice: (FIG. 5A) IgG Fey antibody titers; (FIG. 5B) IgG1 antibody titers;(FIG. 5C) IgG2c antibody titers. Raw data were plotted on Log10 scalewith Box and Whisker Plots (Tukey), boxes=medians with interquartile(IQR) ranges, whiskers=1.5 times the IQR distances. Transformed datawere analyzed by One-way ANOVA with Dunnett post test (compared to G2:CtE nMOMP+Mont+IMO2055), * p<0.05, ** p<0.01, *** p<0.001, NS notsignificant.

FIG. 6 provides results of a murine challenge study. Individual AUCvalues were calculated from qPCR and scatter plotted for each group.Black bar, group median. Data were analyzed by One-way ANOVA (comparedto G1: Mont+IMO2055 adjuvant control), * p<0.05, ** p<0.01, *** p<0.001,NS not significant.

DETAILED DESCRIPTION OF THE INVENTION

Chlamydia major outer membrane protein (MOMP) is a target for vaccinedevelopment to reduce the risk of Chlamydia infection or its associatedclinical manifestations due to its surface exposure and highimmunogenicity. Native MOMP can be purified from an infected cell line,but development of a robust, cost-effective commercial manufacturingprocess based on the use of native MOMP can be challenging. Recombinantexpression of vaccine antigens is an alternative method to purificationof native antigen, which may be easier to scale-up to a commercialmanufacturing level. However, previous attempts to recombinantly expressChlamydia MOMP intracellularly have resulted in insoluble MOMP protein,which is not useful as a vaccine antigen.

To that end, one aspect of the invention provides a method for therecombinant expression of Chlamydia MOMP wherein the MOMP protein isrecombinantly expressed and translocated to the outer membrane of an E.coli cell. Without wishing to be bound by theory, it is thought thatexpression of MOMP in the outer membrane of E. coli results in a MOMPprotein that is folded in a manner that more closely resembles nativeMOMP, which is normally expressed on the cell membrane. Accordingly, themethod of the invention comprises: (a) transforming a population of E.coli host cells with an expression vector comprising a nucleic acidmolecule comprising a sequence of nucleotides that encode a leadersequence for targeting the MOMP to the outer membrane of the cell and asequence of nucleotides that encode Chlamydia MOMP, wherein the nucleicacid molecule is operatively linked to a promoter; (b) culturing thetransformed cells under conditions that permit expression of the nucleicacid molecule and translocation to the outer membrane of the cells toproduce a recombinant Chlamydia MOMP; and (c) optionally purifying theMOMP. The E. coli outer membrane expressed recombinant MOMP produced bythe method of the invention is shown herein to elicit comparableprotection relative to native MOMP in a Chlamydia animal challenge model(see Examples 10-13).

Heterologous protein expression systems may produce inadequateexpression or formation of insoluble protein aggregates due todifferences between the codon usage of the recombinant host cell and thenatural cell type. A “triplet” codon of four possible nucleotide basescan exist in over 60 variant forms. Because these codons provide themessage for only 20 different amino acids (as well as translationinitiation and termination), some amino acids can be coded for by morethan one codon, a phenomenon known as codon redundancy. For reasons notcompletely understood, alternative codons are not uniformly present inthe endogenous DNA of differing types of cells. Indeed, there appears toexist a variable natural hierarchy or “preference” for certain codons incertain types of cells. As one example, the amino acid leucine isspecified by any of six DNA codons including CTA, CTC, CTG, CTT, TTA,and TTG. Exhaustive analysis of genome codon use frequencies formicroorganisms has revealed endogenous DNA of E. coli most commonlycontains the CTG leucine-specifying codon, while the DNA of yeasts andslime molds most commonly includes a TTA leucine-specifying codon. Tothat end, embodiments of the invention provide methods for theheterologous expression of Chlamydia MOMP in the OM of an E. coli hostcell, wherein the gene sequence encoding the MOMP is either (1) codonharmonized or (2) codon optimized for optimal expression in an E. colihost cell.

Thus, in accordance with one aspect of this invention, MOMP-encodinggenes were converted to sequences having identical translated sequencesbut with harmonized codon usage as described by Angov et al.(Heterologous protein expression is enhanced by harmonizing the codonusage frequencies of the target gene with those of the expression host.PLOS one 3(5): 1-10 (2008)). Codon harmonization relies on knownrelationships between secondary protein structure and codon usagefrequencies to modulate translation rates at domain boundaries (i.e.link/end segments). The methodology generally consists of identifyingslowly translated regions in the wild-type mRNA that are associated withdomain boundaries and replacing codons in said region with synonymouscodons having usage frequencies in the recombinant host cell that areless than or equal to the usage frequencies of the codons in the nativeexpression host. Id. For regions outside of the domain boundaries,codons are selected that have usage frequencies that are closely matchedto the native expression system. Id. It is shown herein that expressionof a codon-harmonized DNA sequence encoding Chlamydia MOMP results in ahigher expression level relative to a codon-optimized DNA sequenceencoding the same MOMP polypeptide.

Thus, the invention relates to codon harmonized nucleic acid moleculesencoding Chlamydia MOMP or a derivative thereof, or encoding ChlamydiaMOMP plus a leader sequence for targeting the MOMP to the outer membraneof the cell, as discussed, infra. Also provided by the invention aremethods for the recombinant expression of Chlamydia MOMP, as describedin any embodiment herein, wherein the nucleotide sequence encoding MOMPand/or the nucleotide sequence encoding the leader for targeting theMOMP to the OM of the cell are codon harmonized.

In alternative embodiments of the invention, the MOMP-encoding gene iscodon-optimized for high levels of expression in the intended host cell,e.g. E. coli. The process of codon optimization generally consists ofidentifying codons in the wild-type sequence that are not commonlyassociated with highly expressed genes in the intended host cell andreplacing them with optimal codons for high expression in the intendedhost (i.e. codons that are frequently associated with high levels ofexpression in the recombinant expression host). The new gene sequence isthen inspected for undesired sequences generated by these codonreplacements (e.g., “ATTTA” sequences, inadvertent creation of intronsplice recognition sites, unwanted restriction enzyme sites, high GCcontent, presence of transcription termination signals that arerecognized by yeast, etc.). Undesirable sequences are eliminated bysubstitution of the existing codons with different codons coding for thesame amino acid. The synthetic gene segments are then tested forimproved expression.

The methods described above were used to create synthetic genes encodingMOMP, resulting in a gene comprising codons that are harmonized oroptimized for improved expression in the intended host, e.g. E. coli.While the above procedures provide a summary of the methodology fordesigning codon harmonized and codon-optimized genes for use in themethods of the invention, it is understood by one skilled in the artthat similar expression of genes may be achieved by minor variations inthe procedure or by minor variations in the sequence. For example, theprocedure for codon optimization, as described herein, may comprisereplacement of all of the codons in a given sequence with synonymouscodons associated with high levels of expression in the host cell, orcomprise replacement of only some of the codons in the wild-typesequence, for example, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%or more of the wild-type codons can be replaced. For example, in someinstances, codons in the wild-type sequence may naturally match that ofthe preferred codon in the intended host cell and a replacement with asynonymous codon may not be necessary and/or desired.

As discussed, supra, the methods of the invention utilize an expressionvector comprising a nucleic acid molecule comprising a sequence ofnucleotides that encode a leader sequence for targeting the MOMP to theouter membrane of the cell and a sequence of nucleotides that encodeChlamydia MOMP, wherein the nucleic acid molecule is operatively linkedto a promoter. In embodiments of the invention, the leader sequence isoperatively linked to the N-terminus (i.e. amino or NH₂ terminus) of theMOMP. In preferred embodiments, the leader sequence is directly adjacentto the MOMP sequence. In alternative embodiments, additional amino acidresidues may be present between the leader and the MOMP, e.g. a singleamino acid residue, two amino acid residues, three amino acid residues,four amino acid residues, or five or more amino acid residues. Inembodiments wherein additional amino acid residues are present betweenthe leader and the MOMP, such amino acid residues form a fusion proteinwith the MOMP in the protein product. After expression of the nucleicacid molecule in the E. coli OM, the leader sequence is preferablycleaved from the MOMP protein or MOMP fusion protein, although in somecases, the leader sequence is not cleaved.

As noted above, the methods of the invention are useful for expressionof MOMP fusion proteins in the OM of an E. coli host cell. To that end,in some embodiments the nucleic acid molecule, as described above,further comprises a sequence of nucleotides that encodes an additionalpolypeptide attached to the MOMP, wherein the polypeptide is selectedfrom the group consisting of: a linker, an additional antigen, apolypeptide having adjuvant properties, a polypeptide for facilitatingpurification, a polypeptide for enhancing stability of the MOMP, acarrier protein, and a marker protein.

In embodiments of the invention directed to expression of MOMP fusionproteins in the OM of an E. coli cell, the additional polypeptide can beattached to the N-terminus of the MOMP or the C-terminus of the MOMP. Insome embodiments of the invention, the additional polypeptide isattached to the C-terminus of the MOMP. In additional embodiments, theadditional polypeptide is attached to the N-terminus of the MOMP. Inpreferred embodiments, the nucleic acid molecule comprises, from 5′ to3′, a sequence of nucleotides that encodes a secretion leader fortargeting the protein to the E. coli OM, a sequence of nucleotide thatencodes Chlamydia MOMP, or a derivative thereof, and a sequence ofnucleotides that encodes an additional polypeptide having the attributesdescribed above. However, the invention also contemplates use of nucleicacid molecules that encode, from 5′ to 3′, a leader sequence, anadditional polypeptide, and a MOMP or derivative thereof. One of skillin the art can readily determine whether a MOMP fusion protein made bythe methods of the invention is expressed in the E. coli OM and whetherthe resulting fusion protein has the desired properties by usingprocedures known in the art of molecular and cell biology.

In embodiments of any of the methods of the invention described herein,the MOMP comprises, consists, or consists essentially of the Chlamydiatrachomatis MOMP amino acid sequence set forth in SEQ ID NO:23 (serovarE), SEQ ID NO: 25 (serovar D), SEQ ID NO:26 (serovar G), SEQ ID NO:27(serovar F), SEQ ID NO: 28 (serovar I), SEQ ID NO:29 (serovar J), or SEQID NO:30 (serovar H). In additional embodiments, the MOMP comprises,consists, or consists essentially of the Chlamydia muridarium MOMP aminoacid sequence set forth in SEQ ID NO:31.

In additional embodiments of the invention, the methods compriseexpression of Chlamydia MOMP derivatives in the E. coli OM. In someembodiments, such Chlamydia MOMP derivatives are derivatives of thesequences set forth in SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 or SEQ ID NO:31, whereinthe derivative is at least 85%, at least 90%, at least 95%, at least97%, or at least 99% identical to the reference sequence provided in SEQID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30 or SEQ ID NO:31.

In some embodiments, the MOMP derivative comprises amino acid residuesthat are deleted, inserted or substituted relative to the sequence ofamino acids set forth in the MOMP sequences set forth in SEQ ID NO:23,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30 or SEQ ID NO:31. In particular embodiments, the MOMPderivative comprises a number of amino acid substitutions, deletions oradditions relative to the MOMP sequences disclosed herein, wherein theMOMP derivative comprises no more than 25 amino acid residues, no morethan 20 amino acid residues, no more than 15 amino acid residues, nomore than 12 amino acid residues, no more than 11 amino acid residues,no more than 10 amino acid residues, no more than 9 amino acid residues,no more than 8 amino acid residues, no more than 7 amino acid residues,no more than 6 amino acid residues, no more than 5 amino acid residues,no more than 4 amino acid residues, no more than 3 amino acid residues,no more than 2 amino acid residues, or 1 amino acid residue that is/aresubstituted, deleted or added relative to the MOMP sequences set forthin SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28,SEQ ID NO:29, SEQ ID NO:30 or SEQ ID NO:31. In a particular embodiment,the Chlamydia MOMP derivative is a Chlamydia trachomatis (serovar D)derivative set forth in SEQ ID NO:24, which comprises a 2-amino acidsubstitution relative to SEQ ID NO:25, which was modified in order toincrease Barn-site binding.

In embodiments of any of the methods of the invention, the leadersequence comprises the Shigella flexneri SopA sequence set forth in SEQID NO:8, the Salmonella enterica PgtE sequence set forth in SEQ ID NO:9,the Yersinia pestis Pla set forth in SEQ ID NO:10, the E. coli OmpPsequence set forth in SEQ ID NO:11, the E. coli OmpA sequence set forthin SEQ ID NO:12, or the pectate lysase B (PelB) sequence set forth in orSEQ ID NO:13 or derivative thereof. Thus, in embodiments of theinvention, the leader sequence comprises a sequence of amino acidsselected from the group consisting of: SEQ ID NO:8, SEQ ID NO:9, SEQ IDNO:10, SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13.

In alternative embodiments, the leader sequence comprises a sequence ofamino acids that shares at least 90%, at least 95%, at least 97%, atleast 98%, or at least 99% sequence identity with the amino acidsequence set forth in any of SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQID NO:11, SEQ ID NO:12, or SEQ ID NO:13.

As stated above, the nucleic acid molecule used in the methods of theinvention is operatively linked to a promoter. It is preferable that alow or moderate strength promoter is used in the methods of theinvention. In some embodiments, the promoter is XPL or T7. It is alsopreferred that the expression vector is associated with a rate oftranscription and/or translation that is constrainable to low ormoderate. As used herein a constrainable to low or moderatetranscription/translation rate can result from either elements in thevector itself or elements in the host cell. In some embodiments of theinvention, the expression vector is pAVE029 or pACYDuet-1.

In any of the embodiments of any of the methods of the invention, themethod may further comprise a step of inducing the transformed host cellwith IPTG for from about 4 hours to about 6 hours. In additionalembodiments, the step of inducing with IPTG is carried out for about 3.5hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours,about 6 hours or about 6.5 hours.

In some embodiments of the invention, the induction step described aboveis carried out at about 30° C.

In further embodiments, the cell density (OD590) is allowed to reachabout 0.4 to about 0.8 before the induction step is carried out.

The invention also relates to a recombinant MOMP produced by anyembodiment of any of the methods of the invention. The invention furtherrelates to a recombinant MOMP derivative produced by the methods of theinvention. In some embodiments, the MOMP derivative is a MOMP fusionprotein or chimeric MOMP.

Pharmaceutical Compositions

The invention also relates to pharmaceutical compositions comprising atherapeutically or immunologically effective amount of a recombinantMOMP as described herein, formulated together with a pharmaceuticallyacceptable carrier or diluent.

To prepare pharmaceutical or sterile compositions of the invention, oneor more recombinant Chlamydia MOMP is admixed with a pharmaceuticallyacceptable carrier or excipient. See, e.g., Remington's PharmaceuticalSciences and U.S. Pharmacopeia: National Formulary, Mack PublishingCompany, Easton, Pa. (1984). Pharmaceutically acceptable carriersinclude any and all solvents, dispersion media, isotonic and absorptiondelaying agents, and the like that are physiologically compatible, i.e.suitable for administration to humans. The carriers can be suitable forintravenous, intramuscular, subcutaneous, parenteral, rectal, spinal, orepidermal administration (e.g., by injection or infusion).

As used herein, the term “pharmaceutically acceptable carrier” refers toa substance, as described above, which is admixed with the recombinantMOMP (or derivative) of the invention that is suitable foradministration to humans. In embodiments of the invention, thepharmaceutically acceptable carrier does not occur in nature in the sameform, e.g. the substance is man-made, either because it does not existin nature or the purity and/or sterility of the substance is not thesame as the corresponding natural substance. For example, sterile waterfor injection, which is a sterile, bacteria-free, solute-freepreparation of distilled water for injection, does not occur in naturein the same form and is considered a pharmaceutically acceptablecarrier. In some embodiments, the pharmaceutical compositions of theinvention comprise one or more recombinant MOMP or derivative thereofdisclosed herein and sterile water for injection. In furtherembodiments, the pharmaceutically acceptable carrier may be another formof water that is appropriate for pharmaceutical or biologicalpreparations and is not the same as water that occurs in nature,including purified water, water for injection, sterile purified water,and bacteriostatic water for injection.

In additional embodiments, the pharmaceutical compositions of theinvention include a buffer as a pharmaceutically acceptable carrier.When a buffer is employed, the pH of the buffer is preferably in therange of about 5.5 to about 8.0. In additional embodiments, the pH isabout 5.5 to about 7.5, about 5.5 to about 7.0, about 5.5 to about 6.5,about 6.0 to about 8.0, about 6.0 to about 7.5, about 6.0 to about 7.0,about 6.5 to about 7.0, about 6.0 to 6.5, about 6.0 to about 6.9, about6.2 to about 6.75, or about 6.0 to about 6.75.

Pharmaceutical compositions typically should be sterile and stable underthe conditions of manufacture and storage. Formulations of therapeuticand diagnostic agents may be prepared by mixing with acceptablecarriers, excipients, or stabilizers in the form of, e.g., lyophilizedpowders, slurries, aqueous solutions, suspensions, microemulsions,dispersions, or liposomes, (see, e.g., Hardman, et al. (2001) Goodmanand Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, NewYork, N.Y.; Gennaro (2000) Remington: The Science and Practice ofPharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, etal. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications,Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical DosageForms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weinerand Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc.,New York, N.Y.).

Sterile injectable solutions can be prepared by incorporating the activecompound (i.e., one or more recombinant MOMP and optionally additionalprotein antigen) in the required therapeutically effective amount in anappropriate solvent with one or a combination of ingredients, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients. In the case of sterile powders for the preparation ofsterile injectable solutions, the useful methods of preparation arevacuum drying and freeze-drying that yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof. The proper fluidity of a solution canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prolonged absorption of injectablepharmaceutical compositions can be brought about by including in thepharmaceutical composition an agent that delays absorption, for example,monostearate salts and gelatin. Additional agents, such as polysorbate20 or polysorbate 80, may be added to enhance stability.

Toxicity and therapeutic efficacy of the pharmaceutical compositions ofthe invention, administered alone or in combination with another agent,can be determined by standard pharmaceutical procedures in cell culturesor experimental animals, e.g., for determining the LD₅₀ (the dose lethalto 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index (LD₅₀/ED₅₀). In particularaspects, pharmaceutical compositions exhibiting high therapeutic indicesare desirable. The data obtained from these cell culture assays andanimal studies can be used in formulating a range of dosage for use inhuman. The dosage in such pharmaceutical compositions lies preferablywithin a range of circulating concentrations that include the ED₅₀ withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration.

In some embodiments of the invention, the pharmaceutical compositions ofany embodiment herein can further comprise one or more additional rMOMPantigens produced by the methods of the invention, or derivativethereof, and/or one or more additional non-MOMP Chlamydia antigens. Inspecific embodiments, the pharmaceutical composition comprises, inaddition to a pharmaceutically acceptable carrier, at least onerecombinant MOMP comprising a sequence of amino acids as set forth inSEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27,SEQ ID NO:28, SEQ ID NO:29, or SEQ ID NO:30, or derivative thereof,wherein the rMOMP is produced by the methods of the invention. Inadditional embodiments, the pharmaceutical composition comprises tworMOMP of the invention, or derivatives thereof. In additionalembodiments, the pharmaceutical composition comprises three, four, five,six, seven, eight, or more rMOMP of the invention, or derivativesthereof. The pharmaceutical compositions of the invention can comprisemore than one rMOMP as set forth herein, more than one rMOMP derivative,or a combination of one or more rMOMP of the invention and one or morerMOMP derivative of the invention. In additional embodiments, thepharmaceutical composition comprises at least one rMOMP produced by themethods herein, or derivative thereof, and at least one additionalChlamydia antigen that is not a MOMP or derivative.

In further embodiments of this aspect of the invention, thepharmaceutical compositions comprise one or more recombinant MOMP orderivative thereof produced by the methods disclosed herein, apharmaceutically acceptable carrier, and an adjuvant. The inclusion ofadjuvants may augment the immune response elicited by administration ofthe vaccine antigens (e.g. rMOMP or rMOMP derivative and optionallyadditional Chlamydia antigens) to a patient, in order to induce longlasting protective immunity. In addition to increasing the immuneresponse, adjuvants may be used to decrease the amount of antigennecessary to provoke the desired immune response or decrease the numberof injections needed in a clinical regimen to induce a durable immuneresponse and to provide protection from disease and/or induce regressionof disease caused by Chlamydia infection.

Adjuvants that may be used in conjunction with the pharmaceuticalcompositions of the invention, include, but are not limited to,montanide, adjuvants containing CpG oligonucleotides, or other moleculesacting on toll-like receptors such as TLR4 and TLR9 (for reviews, see,Daubenberger, C. A., Curr. Opin. Mol. Ther. 9(1):45-52 (2007); Duthie etal., Immunological Reviews 239(1): 178-196 (2011); Hedayat et al.,Medicinal Research Reviews 32(2): 294-325 (2012)), includinglipopolysaccharide, monophosphoryl lipid A, and aminoalkyl glucosaminide4-phosphates. Additional adjuvants useful in the pharmaceuticalcompositions of the invention include immunostimulatory oligonucleotides(IMO's; see, e.g. U.S. Pat. Nos. 7,713,535 and 7,470,674, such asIMO-2055, as disclosed in the Examples herein); T-helper epitopes,lipid-A and derivatives or variants thereof, liposomes, calciumphosphate, cytokines, (e.g. granulocyte macrophage-colony stimulatingfactor (GM-CSF) IL-2, IFN-α, Flt-3L), CD40, CD28, CD70, IL-12,heat-shock protein (HSP) 90, CD134 (0X40), CD137, CoVaccine HT,non-ionic block copolymers, incomplete Freund's adjuvant, chemokines,cholera toxin; E. coli heat-labile enterotoxin; pertussis toxin; muramyldipeptide, muramyl peptide analogues, MF59, SAF, immunostimulatorycomplexes, biodegradable microspheres, polyphosphazene; andpolynucleotides.

Additional adjuvants for use with the pharmaceutical compositionsdescribed herein are adjuvants containing saponins (e.g. QS21), eitheralone or combined with cholesterol and phospholipid in thecharacteristic form of an ISCOM (“immune stimulating complex,” forreview, see Barr and Mitchell, Immunology and Cell Biology 74: 8-25(1996); and Skene and Sutton, Methods 40: 53-59 (2006)). Such adjuvantsare referred to herein as “saponin-based adjuvants”. In specificembodiments of the pharmaceutical compositions and methods providedherein, the recombinant MOMP antigens are combined with an ISCOM-typeadjuvant or “ISCOM”, which is an ISCOM matrix particle adjuvant, such asISCOMATRIX™, which is manufactured without antigen (ISCOM™ andISCOMATRIX™ are the registered trademarks of CSL Limited, Parkville,Australia).

Additionally, aluminum-based compounds, such as aluminum hydroxide(Al(OH)₃), aluminum hydroxyphosphate (AlPO₄), amorphous aluminumhydroxyphosphate sulfate (AAHS) or so-called “alum” (KAl (SO₄)·12H₂O)(see Klein et al., Analysis of aluminum hydroxyphosphate vaccineadjuvants by Al MAS NMR., J. Pharm. Sci. 89(3): 311-21 (2000)), may becombined with the pharmaceutical compositions provided herein.

In some embodiments described herein, the adjuvant is an aluminum saltadjuvant. In alternative embodiments, the adjuvant is a saponin-basedadjuvant or a toll-like receptor agonist adjuvant. In exemplaryembodiments of the invention provided herein, the aluminum adjuvant isaluminum hydroxyphosphate or AAHS, alternatively referred to as “MAA”.In alternative embodiments, the adjuvant is aluminum hydroxide. Infurther embodiments, the adjuvant is aluminum phosphate.

Adjuvants may be combined to provoke the desired immune response. Forexample, the pharmaceutical compositions of the invention may compriseat least one rMOMP produced by the methods described herein, apharmaceutically acceptable carrier and a combination of two or moreadjuvants. In some embodiments of the invention, the pharmaceuticalcompositions comprise an aluminum salt adjuvant and a second adjuvant.In other embodiments, the compositions comprise an aluminum saltadjuvant and a second adjuvant selected from a saponin adjuvant and atoll-like receptor agonist.

Methods of Use

Embodiments of the invention also include one or more of the recombinantMOMP, or derivative thereof, or pharmaceutical compositions comprisingsaid recombinant MOMP or derivative, or a vaccine comprising saidrecombinant MOMP or pharmaceutical compositions (i) for use in, (ii) foruse as a medicament or composition for, or (iii) for use in thepreparation of a medicament for: (a) therapy (e.g., of the human body);(b) medicine; (c) inhibition of Chlamydia replication; (d) treatment orprophylaxis of infection by Chlamydia; (e) prevention of recurrence ofChlamydia infection; (f) reduction of the progression, onset or severityof pathological symptoms associated with Chlamydia infection and/orreduction of the likelihood of a Chlamydia infection or, (g) treatment,prophylaxis of, or delay in the onset, severity, or progression ofChlamydia-associated disease(s), including, but not limited to:oculogenital disease, cervicitis, urethritis, endometritis, pelvicinflammatory disease, tubal infertility, ectopic pregnancy, neonatalconjunctivitis, and infant pneumonia. In the uses set forth herein, therecombinant MOMP or derivatives thereof, pharmaceutical compositionsand/or vaccines comprising or consisting of said recombinant MOMP canoptionally be employed in combination with one or more additionaltherapeutic agents, for example, a second vaccine for a differentpathogen.

Thus, the invention relates to a method as set forth above, which methodcomprises administration of an immunologically or therapeuticallyeffective amount of any rMOMP or rMOMP derivative produced by themethods of the invention, or pharmaceutical composition or vaccinethereof, to a patient in need thereof, whereby administration to thepatient results in any of (c) through (g) above.

The mode of administration to the patient can vary and may include oral,rectal, transmucosal, intestinal, parenteral; intramuscular,subcutaneous, intradermal, intrathecal, direct intraventricular,intravenous, intraperitoneal, intranasal, intraocular, inhalation,insufflation, topical, cutaneous, transdermal, or intra-arterial. Inembodiments of the invention, the route of administration is parenteral.In specific embodiments of the invention, the mode of administration issubcutaneous or intraperitoneal. In additional embodiments of theinvention, the route of administration is intramuscular, intradermal, orsubcutaneous.

The rMOMP, derivatives, or pharmaceutical compositions of the inventioncan be administered with medical devices known in the art. For example,a pharmaceutical composition of the invention can be administered byinjection with a hypodermic needle, including, e.g., a prefilled syringeor autoinjector.

The pharmaceutical compositions disclosed herein may also beadministered with a needleless hypodermic injection device; such as thedevices disclosed in U.S. Pat. Nos. 6,620,135; 6,096,002; 5,399,163;5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.

Codon-Harmonized MOMP Nucleotide Sequences

One aspect of the invention relates to codon harmonized nucleic acidmolecules encoding Chlamydia MOMP or a derivative thereof, or encodingChlamydia MOMP plus a leader sequence for targeting the MOMP to theouter membrane of the cell. In particular embodiments, the inventionprovides a nucleic acid molecule that encodes the Chlamydia trachomatisMOMP set forth in SEQ ID NO:23, SEQ ID NO: 25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO: 28, SEQ ID NO:29, SEQ ID NO:30, wherein the nucleicacid molecule is codon-harmonized. The invention also provides acodon-harmonized nucleic acid molecule that encodes the Chlamydiamuridarium MOMP set forth in SEQ ID NO:31.

In additional embodiments, the invention provides codon-harmonizednucleic acid molecules that encode variants or derivatives of theChlamydia MOMP amino acid sequences described herein. Thus, theinvention relates to codon-harmonized nucleic acid molecules encodingderivatives of the Chlamydia MOMP sequences set forth in SEQ ID NO:23,SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29,SEQ ID NO:30 or SEQ ID NO:31, wherein the derivative is at least 85%, atleast 90%, at least 95%, at least 97%, or at least 99% identical to thereference sequence provided in SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:26,SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 or SEQ ID NO:31.

In some embodiments, the codon-harmonized nucleic acid molecule encodesa MOMP derivative that comprises amino acid residues that are deleted,inserted or substituted relative to the sequence of amino acids setforth in the MOMP sequences set forth in SEQ ID NO:23, SEQ ID NO:25, SEQID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30 or SEQID NO:31. In particular embodiments, the MOMP derivative comprises anumber of amino acid substitutions, deletions or additions relative tothe MOMP sequences disclosed herein, wherein the MOMP derivativecomprises no more than 25 amino acid residues, no more than 20 aminoacid residues, no more than 15 amino acid residues, no more than 12amino acid residues, no more than 11 amino acid residues, no more than10 amino acid residues, no more than 9 amino acid residues, no more than8 amino acid residues, no more than 7 amino acid residues, no more than6 amino acid residues, no more than 5 amino acid residues, no more than4 amino acid residues, no more than 3 amino acid residues, no more than2 amino acid residues, or 1 amino acid residue that is/are substituted,deleted or added relative to the MOMP sequences set forth in SEQ IDNO:23, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30 or SEQ ID NO:31. In a particular embodiment, thecodon-harmonized nucleic acid molecule encodes the Chlamydia trachomatis(serovar D) derivative set forth in SEQ ID NO:24, which comprises a2-amino acid substitution relative to SEQ ID NO:25, which was modifiedin order to increase Barn-site binding.

The invention further relates to nucleic acid molecules that encode aChlamydia MOMP polypeptide and a leader sequence for targeting the MOMPto the OM of the cell, wherein the nucleic acid molecules arecodon-harmonized.

In some embodiments, the Chlamydia MOMP is any MOMP polypeptide sequenceor MOMP derivative polypeptide sequence disclosed herein, and the leadersequence comprises the Shigella flexneri SopA sequence set forth in SEQID NO:8, the Salmonella enterica PgtE sequence set forth in SEQ ID NO:9,the Yersinia pestis Pla set forth in SEQ ID NO:10, the E. coli OmpPsequence set forth in SEQ ID NO:11, the E. coli OmpA sequence set forthin SEQ ID NO:12, or the pectate lysase B (PelB) sequence set forth in orSEQ ID NO:13. In particular embodiments, the codon-harmonized nucleicacid molecule encodes a Chlamydia MOMP+leader sequence set forth in SEQID NO:16, SEQ ID NO:19, or SEQ ID NO:22. In additional particularembodiments, the nucleic acid molecule that encodes the ChlamydiaMOMP+leader sequence comprises a sequence of nucleic acids as set forthin SEQ ID NO:15, SEQ ID NO:18, or SEQ ID NO:21, or a nucleic acidderivative thereof.

All publications mentioned herein are incorporated by reference for thepurpose of describing and disclosing methodologies and materials thatmight be used in connection with the present invention.

Having described preferred embodiments of the invention with referenceto the accompanying drawings, it is to be understood that the inventionis not limited to those precise embodiments, and that various changesand modifications may be effected therein by one skilled in the artwithout departing from the scope or spirit of the invention as definedin the appended claims.

EXAMPLE 1 Expression of Recombinant Chlamydia MOMP A. CodonHarmonization

β-barrel membrane protein expression directed to the E. coli outermembrane (OM expression) is typically challenging. We evaluated theeffect of codon selection on the surface expression of full lengthrecombinant Chlamydia MOMP (rMOMP). We performed codon harmonization andstandard codon optimization on the recombinant MOMP gene and evaluatedouter membrane expression with a whole cell flow cytometry binding assayusing anti-Chlamydial EB mouse sera. Both genes were expressed in a pETvector expression system and with a native Chlamydia muridarium (Cm)MOMP leader sequence (Tables 2 and 3). We observed that codonharmonization resulted in -2 fold increase in outer membrane expressionof recombinant Chlamydia muridarium MOMP, compared to the standard hostcodon optimized gene (FIG. 1 ), suggesting that better protein foldingand OM expression were achieved with the codon harmonized generegulating the rate of translation. Upon this finding, codon harmonizedrMOMP genes have been used in subsequent expression evaluations (such asoptimal expression vectors and leader sequences) to further improverMOMP OM expression.

B. Expression Vector Optimization

A panel of E. coli expression vectors were evaluated to further increasethe surface expression level of rMOMP (Table 2). The key elements thatcould affect the OM expression include promoter strength and vector copynumber. We compared vectors with high, medium or low copy numbers, withpromoters of high, moderate or titratable strength. We found that eithera strong promoter or a high vector copy number limited the surfaceexpression of rMOMP (Table 2). Higher rMOMP surface expression wasachieved with a combination of moderate promoter and a low vector copynumber (such as pAVE029), suggesting that lower transcription level ispreferred. Consistently, reasonable rMOMP OM expression level can beobtained with a pACYDuet vector when we used a host strain with acontrollable RNA polymerase level to reduce the rMOMP mRNA transcriptionrate. We hypothesized that slower transcription and therefore slowertranslation is optimal for rMOMP OM expression as it provides ample timeto allow the newly synthesized protein to properly fold and translocateonto the outer membrane, resulting in an increased level of surfaceexpression. This result is also consistent with our observations on theeffect of gene codon usage harmonization described above.

TABLE 2 Evaluation of E. coli expression vectors Origin of Copy rMOMPSurface Vector Promoter/Strength Inducer Replication Number Expression(GFI*) **pAVE029 λPL/Moderate IPTG pAT153(colE1) Low Good (~300)pACYDuet-1 T7/Titratable Arabinose + p15A Low Intermediate (~120) IPTGpET (pETBlue-1 T7/Strong IPTG pUC High Low(~30 or lower) and pET22b)pWSK29 T7/T3/Strong IPTG pSC101 Low None (intracellular) pJ831(pUC)T7/Titratable Rhamnose pMB1 High None (intracellular) pJ841(pBR)T7/Titratable Rhamnose pMB1 Medium None (intracellular) pJ851(pACYC)T7/Titratable Rhamnose p15A Low None (intracellular) *GFI: geomeanfluorescence intensity from whole cell flow cytometry binding assay**pAVE029 is an E. coli RNA polymerase dependent expression vector.Others listed are bacteriophage T7 RNA polymerase dependent expressionvectors.

C. Secretion Leader Sequence Optimization

In order to better direct the OM localization of rMOMP, we evaluateddifferent secretion leader sequences which might help the OMlocalization of the target protein (Table 3). Among tested leadersequences, E. coli OmpA leader and OmpP leader resulted in the highestrMOMP OM expression. However, incomplete cleavage of the OmpP leader wasobserved and heterologous forms of rMOMP were generated (data notshown). Omptins leader family and PelB leader resulted in similar levelsof moderate surface expression of rMOMP. Native Cm MOMP leader is ableto direct the OM expression for CmMOMP, but not for CtD or CtE MOMP.Neither native CtD or CtE MOMP leaders result in the surface expressionof rMOMP.

TABLE 3 rMOMP Secretion Surface Leader Secretion Expression CtD_Sequences Leader Cm_ CT_ CtE_ Evaluated Sequences MOMP MOMP MOMP NativeMKKLLKSVLAF + − − Cm MOMP AVLGSASSLHA (SEQ ID NO: 6) Native MKKLLKSVLVFND − − CtD/CtE AALGSASSLQA MOMP (SEQ ID NO: 7) *Shigella MKSKFLVLALC NDND + flexneri VPAIFTTHA (SopA) (SEQ ID NO: 8) *salmonella  MKTHVIAVMIIND ND + enterica AVFSESVYA (PgtE) (SEQ ID NO: 9) *Yersinia MKKSSIVATIIND ND + pestis (Pla) TILSGSANA (SEQ ID NO: 10) E.coli. MQTKLLAIMLA ND ND++ OmpP APVVFSSQEASA (SEQ ID NO: 11) E.coli. MKKTAIAIAVA + ND ++ OmpALAGFATVAQA (SEQ ID NO: 12) pectate MKYLLPTAAAG + + + lyase B ofLLLLAAQPAMA Erwinia (SEQ ID NO: 13) carotovora CE (PelB) *Belongs toomptins leader sequence family (trans membrane aspartic proteases) ND:Not Determined Cm: C. muridarium CtD_CT: C. trachomatis Serovar D (Cterminal AA sequence modified) CtE: C. trachomatis SerovarE

D. Optimization of Expression Conditions

We evaluated many expression conditions with the pAVE029 expressionsystem that could impact rMOMP OM expression, including cell culturemedium, cell density at induction, induction time and temperature (FIG.2 ). rCtE MOMP with a PelB leader sequence was used in this evaluation.We performed induction for 4 hrs, 6 hrs and 16 hrs under four differenttemperatures: 16° C., 25° C., 30° C. and 37° C. We found that 4 hrs and6 hrs of IPTG induction resulted in comparable rMOMP OM expressionlevels while no expression was observed for 16 hrs induction at any ofthe temperatures tested (FIG. 2A). Induction at 37° C. or 30° C.resulted in high surface expression of rMOMP (FIG. 2A). However, weobserved cell fragility at 37° C., indicated by the decreased OD590after induction (FIG. 2B). We also performed induction at different celldensities and found that it dramatically impacts the rMOMP surfaceexpression. We obtained the highest rMOMP expression with an inductionOD590 of ˜0.5, while expression dropped with an induction OD590 of ˜0.8,and little or no expression with an induction OD590 of 1.2 or higher(FIG. 2C).

Seven different cell culture media were evaluated for their effect onexpression level: 1-LB (Luria broth), 2-2YT (2× yeast extract andtryptone) +1% Glu, 3-Mg, 4-ProGro™ (Expression Technologies, Inc., SanDiego, Calif.), 5- Azura, 6-Cinnabar, and 7-Auto induction medium).Interestingly, we observed very different rMOMP OM expression (FIG. 2D).We obtained very high level of periplasmic rMOMP expression (data notshown) with Progro medium, however, no surface expression was observed.Low levels of rMOMP OM expression were observed with LB medium, 0.2%lactose auto induction medium, 2YT medium with 1% glucose, and thechemically defined Azura medium. The highest rMOMP OM expression (GFI˜300 to 400 in the whole cell flow cytometry binding assay) was obtainedwith growth in Cinnabar medium, resulting in a visible rMOMP band on aSDS-PAGE gel. IPTG concentration for induction was also evaluated andcomparable rMOMP OM expression was observed with 0.1 mM to 1 mM IPTG(data not shown). Therefore, expression studies described herein wereinduced by addition of 0.4 mM IPTG.

In summary, the most optimized conditions we have obtained for rMOMP OMexpression is to perform induction for 4 hrs at 30° C. when cell density(OD590) reaches ˜0.5 (FIG. 3 ). The optimal expression level isincreased ˜10 fold compared to the original expression conditions. Threedifferent rMOMP proteins (Cm, CtD and CtE) have been successfullyexpressed on E. coli outer membrane under these conditions.

EXAMPLE 2 Purification of Recombinant Chlamydia MOMP

Harvested E. coli cells expressing recombinant MOMP were disrupted bymicrofluidization and membrane fraction containing rMOMP was pelleted byultra-centrifugation, while soluble cellular proteins were largelyseparated. Washing the membrane fraction with high salt buffer furtherremoved residual soluble cellular proteins. Subsequent wash with abuffer containing 1% triton X-100 detergent removed the bacterial innermembrane and wash with a buffer containing 3% beta-octyl-glucosidedetergent removed certain bacterial outer membrane proteins other thanrecombinant MOMP. After these steps, rMOMP became the most abundantprotein in the membrane fraction. A variety of detergents were evaluatedfor extraction of rMOMP from the outer membrane. We found that sarkosyl(an anionic detergent) was the most efficient, followed by foscholine-14(a lipid like zwitterionic detergent) and zwittergent 3-12. DTT wasrequired for extraction of rMOMP. The extraction contained ˜60% rMOMP.Extracted rMOMP was further purified by size exclusion and ion exchangechromatography. The purified rMOMP migrates very similarly to the nativeMOMP (nMOMP) protein that was purified from Chlamydia elementary body(EB) on a SDS-PAGE gel, with slightly higher amounts of dimeric andoligomeric forms (FIG. 4A and 4B). In-solution mass spectrometrysuggested that final purified rCtE-MOMP is about 70% pure with a fewco-purified E. coli proteins (FIG. 4C). The endotoxin level in the finalpurified protein sample is undetectable with an anti-E. coli LPS mAb onwestern blot (FIG. 4D) or in the LAL assay (data not shown).

EXAMPLE 3 Mouse Immunogenicity and Challenge Study

Female C57BL/6 mice (32 per group) were immunized by subcutaneous (s.c.)routes with purified nMOMP or rMOMP (10m/mouse/immunization) incombination with an adjuvant containing IMO-2055 and Montanide ISA 720VG. Two preparations of rCtE-MOMP were evaluated: one with a PelB leadersequence and the other one with an OmpA leader sequence. A positivecontrol group was immunized with 1×10⁶ live EB in SPG per mouse byintraperitoneal (i.p.) route. A negative control group (adjuvantcontrol) was administered with a combination of IMO-2055 and MontanideISA 720 VG only.

Post-immunization mouse serum was analyzed by ELISA with CtD EBs as thecoating antigen (FIG. 5 ). In general, the rMOMP immunized mice groupstended to have more variability in titers, compared to nMOMP immunizedmice group. The PelB-leader-rMOMP immunized mice have comparable (nostatistical different) IgGFcγ titers and slightly lower IgG1 and IgG2ctiters compared to the nMOMP immunized group. The OmpA-leader-rMOMPimmunized mice have lower IgGFcγ, IgG1 and IgG2c titers than the nMOMPimmunized group.

Two weeks following the last immunization, mice were challengedintravaginally with CtD EBs. The vaginal vault and ectocervix wereswabbed on multiple time points following challenge and Chlamydia copynumbers were evaluated by real-time PCR. For each animal, qPCR valueswere log transformed at each time point and AUC (area under curve) valuewas calculated for the time period (FIG. 6 ). Both preparations of therCtE-MOMP (with a PelB leader or with an OmpA leader) elicitedstatistical significant protective responses (p<0.01) in immunized mice,compared to the group that received the adjuvant only (FIG. 6 ).

Moreover, the level of protection from bacterial challenge in miceimmunized with the PelB-leader-rMOMP was comparable (similar bacterialload post challenge) to the level in nMOMP immunized group. The level ofprotection from bacterial challenge in the OmpA-leader-rMOMP immunizedmice was slightly lower (higher bacterial load post challenge) than thelevel in nMOMP immunized group, consistent with the lower overall IgGantibody titers induced in this group.

Our data suggested that the recombinant MOMP expressed on and purifiedfrom the outer membrane of E. coli elicits protective serum antibodyresponses in a mouse challenge model, therefore, can be evaluated as apotential candidate for a vaccine against Chlamydia.

Materials and Methods EXAMPLE 4 Codon Harmonization of the ChlamydiaMOMP Gene for Recombinant Expression

Nucleotide sequences of the gene encoding the Major Outer MembraneProtein (MOMP) were retrieved from Merck internal website CMR(Comprehensive Microbial Resources) for the following strains: C.muridarum Nigg (strain MoPn) ORF TC0052 (GenBank Gene ID: 1245581;Protein Accession No. P75024.1); C. trachomatis strain D/UW-3/CX CT ORFTC_681 Serovar D (GenBank Gene ID: 884473; Protein Accession No.NP_220200.1); and C. trachomatis strain E/12-94 ORF 0175_03780 Serovar E(GenBank Gene ID: 16635280; Protein Accession No. P17451). Amino acidsequences consisting of a secretion leader and the mature MOMP protein(Table 3) were codon harmonized (Angov et al., Plos One 3(5):1-10(2008)). In brief, the codon usage data for Chlamydia (native host) andE. coli (expression host) was obtained from the Codon Usage Database(tabulated from NCBI-Genbank, Kazusa DNA Res. Inst., Kisarazu, Japan).For each species, the strain with the most codon usage data availablewas selected as a representative. The codon usage frequency for bothnative and expression hosts was then calculated and a reference databasewas generated. We first identified the amino acid residues for which therare codons were used in the native host, and the corresponding rarecodon in the expression host was selected for those residues. For theremaining residues, the codon in the expression host that has theclosest frequency (less than 15% difference) to the corresponding codonin the native host was selected. If a codon in E. coli could not beidentified that had less than 15% difference relative to the frequencyof the native codon, a codon with 15% or more lower frequency was chosenif the residue was in a “linker/hinge” region in order to slow down thetranslation speed and a codon with 15% or more higher frequency wasselected if the residue was outside the linker region to achieve higherexpression. Once the harmonized gene sequence for Chlamydia MOMP wasgenerated, NdeI and XhoI restriction enzyme sites were mutated forsubsequent cloning.

EXAMPLE 5 Cloning and Expression of Recombinant Chlamydia MOMP

The harmonized gene sequences with flanking NdeI and XhoI restrictionenzyme sites were synthesized and cloned into the PUC57 cloning vector.The synthesized genes were excised from PUC57 vector through NdeI andXhoI restriction sites. The excised DNA fragments were ligated into thepAVE029 expression vector (MSD Biologics UK) using T4 DNA ligase(Promega Corp., Madison, Wis.) for 4 hours at 16° C. Ligated plasmidswere transformed into competent cells DH5a (Invitrogen, Carlsbad,Calif.) and grown in LB agar plates with 10 μg/mL tetracycline. Coloniesharboring the recombinant plasmid were identified by PCR and confirmedby sequencing using pAVE029 vector specific primers for 5′ end of thegene (ppop40 primer ATT CTG CAT TCA CTG GCC GAG G (SEQ ID NO:1)) and 3′end of the gene (T7 Term standard sequencing primer GCT AGT TAT TGC TCAGCG G (SEQ ID

NO:2)). The sequence-confirmed positive colonies were propagated in LBmedium with 10 μg/mL of tetracycline and plasmid DNA was isolated fromthe cell cultures with a HiSpeed Maxi Kit (QIAGEN, Venlo, Netherlands).

The recombinant plasmid DNA was transformed by electroporation into anexpression host strain E. coli K12 W25113 using a Bio-Rad GenePulser(Bio-Rad Laboratories, Inc., Hercules, Calif.. Transformed cells wereplated on LB Agar plates with 10 μg/mL tetracycline and grown overnightat 37° C. Single colonies were picked and inoculated into Cinnabar media(Teknova, Hollister, Calif.) with 10 μg/mL of tetracycline and grown at37° C. with shaking at 250 RPM until OD600 reaches to mid log phase(˜0.5). 0.4 mM IPTG was added into the cell culture for induction andthe cell culture was incubated for 4 hours at 30° C. with shaking.

The cell cultures were then characterized by whole cell flow cytometrybinding assay, SDS-PAGE, and Western Blot analysis.

EXAMPLE 6 Whole Cell Flow Cytometry Binding

50 μL of E. coli cell culture (at ˜1×10⁹ cells/mL) that recombinantlyexpressed Chlamydia MOMP was incubated with 50 μL of mouse sera againstChlamydia elementary body (EB) at a dilution of 1:250 for 1 hour at roomtemperature in a 96 well plate. After incubation, the cells were washedwith 1 mL phosphate buffered saline (PBS) and stained with 100 μL of afluorescence labeled secondary antibody (Alexa Fluo-488 F(ab)′2 fragmentof goat anti-mouse IgG (H+L), Life Technologies, Carlsbad, Calif.) at adilution of 1:100. The stained cells were washed twice and re-suspendedin PBS for flow cytometric analysis (Guava Technologies, EMD Millipore,Billerica, Mass.). Data analyses were performed with CytoSoft 5.3software (Guava Technologies).

EXAMPLE 7 SDS-PAGE and Western Blot

E. coli cell culture (˜1×10⁹ cells) that recombinantly expressesChlamydia MOMP was treated with SDS loading buffer with reducing agent(Invitrogen, Carlsbad, Calif.). Samples were applied to NuPAGE(Invitrogen) gel electrophoresis. NuPAGE gel was stained with Gel codeblue staining solution (Pierce Biotechnology, Rockford, Ill.). ForWestern Blot, samples were applied to gel electrophoresis and thenelectro-transferred onto nitrocellulose membranes (Life Technologies,Carlsbad, Calif.). The membranes were incubated with mouse sera againstChlamydia EB (or other specific primary antibodies) followed by afluorescence conjugated goat anti-mouse secondary antibody (IRDye 680LT,Licor). Image was acquired and analyzed by LICOR ODYSSEY® (Li-CorBiosciences, Lincoln Nebr.).

EXAMPLE 8 Purification of Recombinant Chlamydia MOMP

E. coli cell culture was grown in Cinnabar media (Teknova, Hollister,Calif.) and induced by IPTG as described above. Cell culture washarvested by centrifugation at 12,000 g for 15 min. Cell pellets wereweighed and resuspended in 9 volumes (v/w) of 50 mM Tris-Cl pH 8.0buffer with EDTA free protease inhibitor (Roche, Basel, Switzerland, 1tab per 100 mL buffer). Cells were disrupted by microfluidization andundisrupted cells were pelleted and removed by centrifugation at 9700 gfor 15 min. Membrane fraction was pelleted by centrifugation of thecleared disrupted cells at 23800 g for 90 min and washed with high saltbuffer (1M NaCl, 0.05% tween20) followed by another centrifugation at23800 g for 90 min. To remove the bacterial inner membrane, washedmembrane fraction was resuspended in buffer A (20 mM Tris-Cl pH 8.0, 1mM EDTA) with 1% triton X-100, incubated at room temperature for 15 minfollowed by ultracentrifugation at 120,000 g for 40 min. To removebacterial outer membrane proteins other than recombinant MOMP (rMOMP),pellets were resuspended by buffer A (20 mM Tris-Cl pH 8.0, 1 mM EDTA)with 3% beta-octyl-glucoside, incubated at room temperature for 1 hourfollowed by ultracentrifugation at 120,000 g for 40 min. rMOMP wasextracted by resuspending the pellets in buffer A (20 mM Tris-Cl pH 8.0,1 mM EDTA) with 1% sarkosyl, incubated at room temperature for 2 hourfollowed by ultracentrifugation at 120,000 g for 40 min. Extracted rMOMPwas subjected to size exclusion chromatography (Sephacryl S300, GEhealthcare) in a buffer containing 10 mM Hepes pH 7.3, 150 mM NaCl, 0.1%zwittergent 3-14. Eluted rMOMP was further purified with ion exchangechromatography (Hitrap Q FF, GE healthcare). Purified rMOMP fractionswere pooled and stored at 4° C.

EXAMPLE 9 Cell Culture and Propagation of Chlamydiae

All cell lines and Chlamydia strains were obtained from ATCC (Manassas,Va.). HeLa 229 cells were used for propagation all strains. HeLa 229cells were grown in Eagle's Minimal Essential Medium (ATCC) supplementedwith 10% heat-inactivated fetal bovine serum (FBS, Hyclone), 50 μg/mLvancomycin (Sigma-Aldrich, St. Louis, Mo.), and 10 μg/mL gentamicin(Gibco). Host cells were seeded into tissue culture flasks at a celldensity of 5×10⁵ cells/mL and incubated overnight at 37° C. in 5% CO₂ toachieve a confluent monolayer. Cell monolayers were infected with C.trachomatis (Ct) strain D/UW-2/Cx stock diluted insucrose-phosphate-glutamate (SPG) buffer and cultured for 72 hours. TheChlamydiae were harvested from the infected cells and purified bycentrifugation through 30% Renograffin (Bracco Diagnostics, Milan Italy)and stored frozen at ˜80° C.

EXAMPLE 1 Mouse Immunization

Female C57BL/6 mice (Taconic Farms) were used at 6 to 8 weeks of age,and food and water were provided ad libitum. All animal procedures werein accordance with government and institutional guidelines for animalhealth and well-being, and were approved by the Merck InstitutionalAnimal Care and Use Committee.

Animals were immunized by subcutaneous (s.c.) routes with rMOMP (1 to 10μg/mouse/immunization) in combination with an adjuvant containingIMO-2055 and Montanide ISA 720 VG (SEPPIC Inc., Coley PharmaceuticalGroup Inc., Wellesley, Mass.) at a ratio of 70:30 (v/v). Live EB groupswere immunized with 1×10⁶ EB in SPG per mouse by intraperitoneal (i.p.)route. Adjuvant control groups were administered with a combination ofIMO-2055 and Montanide ISA 720 VG only. Immunizations were administeredon days 0, 20 and 30.

Prior to the first immunization and two weeks following the finalimmunization, tail bleeds were performed with blood collected in BDMicrotainer® Serum Separator Tubes (Becton, Dickinson and Company,Franklin Lakes, N.J.). Blood samples were centrifuged at 6,000 rpm for 5min and serum was transferred to a microcentrifuge tube.

EXAMPLE 11 Murine Challenge Study

At approximately 2 weeks following the last immunization, progesterone(medroxyprogesterone acetate, Depo-Provera; Pfizer, New York, N.Y.) wasadministered subcutaneously (2.5 mg/dose) at 10 and 3 days beforechallenge. Mice were challenged intravaginally (approximately 1 monthfollowing the last immunization) by direct instillation of 10 μL of SPGcontaining 1×10⁵ Ct serovar D EBs. The vaginal vault and ectocervix wereswabbed using a microfiber swab (Fisher, Hampton, N.H.) on days 7, 11,14, 18, and 21 (or a combination of these time points) followingchallenge.

Swabs were placed into a 1.5-mL tube containing 2 sterile glass beads (5mm diameter) and 300 μL of Chlamydia isolation medium (Trinity Biotech,Bray, Ireland) on ice. Bacteria were eluted from the swabs and separatedfrom cells by vortexing for 60 seconds. 100 μL of eluted cells/bacteriawere plated onto a processing cartridge containing 100 μL of PBS andstored at −70° C. until DNA extraction.

EXAMPLE 12 Primer, Probe and Real-Time PCR

DNA from genital swab samples was extracted using the MagNA Pure 96 DNAand Viral NA small volume kit (Roche, Basel, Switzerland) on the MagNApure machine (Roche) according to the manufacturer's instructions.

The oligonucleotide primer set was designed for detection of all speciesof Chlamydiae. The sense primer, 16S DIR 5′-CGC CTG AGG AGT ACA CTCGC-3′ (SEQ ID NO:3), and anti-sense primer, 16S Rev 5′-CCA ACA CCT CACGGC ACG AG-3′ (SEQ ID NO:4), were designed to amplify a 208-bp fragmentof the chlamydial 16S ribosomal subunit gene, conserved across Chlamydiastrains and serovars. Primers were obtained from Sigma Genosys (TheWoodlands, TX), and the probe, 16S Fam-5′-CAC AAG CAG TGG AGC ATG TGGTTT AA-3′ Tamra (SEQ ID NO:5), was synthesized by Applied Biosystems,(Foster City, Calif.).

The 50-μL reaction mixtures consisted of 1× QuantiTect Multiplex PCRmaster mix without ROX (Qiagen, venlo, netherlands), 100 nmol/L 16Sprobe, 200 nmol/L primer 16S DIR, 400 nmol/L primer 16S Rev, 30 nmol/LROX reference dye, and 5 μL of sample DNA. Nontemplate controlsconsisting of the reaction master mix, primers, and probe, but no DNA,were included in each assay run. Reaction conditions were set asfollows: 1 cycle at 95° C. for 15 min, followed by 40 cycles at 94° C.for 1 min and at 60° C. for 1 min. Thermal cycling, fluorescent datacollection, and data analysis were performed using the StratageneMx3005P system (Stratagene, La Jolla, Calif.) according to themanufacturer's instructions.

EXAMPLE 13 Detection of Serum Antibody and Isotype Levels by ELISA

Serum was analyzed by an enzyme-linked immunosorbent assay (ELISA). Nunc™C96 Maxisorp Immunoplates (Thermo Scientific, Waltham, Mass.) werecoated with 50 uL of 1 ug/ml C. trachomatis Serovar D EBs in PBS andrefrigerated overnight. The plates were washed three times with 0.05%Tween-20 (Fisher Scientific) in PBS (PBS-T). The wells were blocked with5% HyClone® Fetal Bovine Serum (FBS) (Thermo Scientific) in PBS at 200μL/well for 1 hour at room temperature and washed three times withPBS-T. Serum was diluted in 5% FBS in PBS at a 1:500 dilution. Seriallydiluted sera were added to the plate, incubated for 2 hours at roomtemperature and the plates were washed three times with PBS-T.HRP-conjugated secondary antibodies (Goat anti-mouse IgG, Fcγ fragmentspecific; Goat Anti-mouse IgG, Fcγ Subclass 1 specific; or GoatAnti-mouse IgG, Fcγ Subclass 2c specific; Jackson ImmunoResearchLaboratories, Inc., West Grove, Pa.) were diluted in 5% FBS in PBS at1:6,000, 1:6,000, or 1:2,000 dilution, respectively. The dilutedsecondary antibodies were added at 100 μL/well, incubated for 1 hour atroom temperature and the plates were washed three times with PBS-Tfollowed by three times with PBS. Room temperature BD Opt EIA™ TMBSubstrate Reagent Set (BD Biosciences, Franklin Lakes, N.J.) was mixedand filtered through a 0.22 um CA filter unit (Corning, Inc., CorningNY), and 100 μL was added to each well and incubated for 10 min at roomtemperature. The reaction was stopped with 100 μL/well of 2M H₂SO₄(Fisher Scientific). The optical density (OD) was read at 450 nm on aSpectraMax® M5 (Molecular Devices). The cutoff OD for eachpost-imunization serum was calculated as two times of the OD₄₅₀ of thecorresponding pre-immunization serum. ELISA titers were determined bylinearly interpolating between the sequential log dilutions that bracketthe cutoff OD, where the dependent variable is the OD response and theindependent variable is the log dilution. The resulting dilution is thenback transformed to obtain the reported titer. The reported titer is theestimated dilution of serum that results in a response equivalent to thecutoff OD.

What is claimed is:
 1. A method for the recombinant expression ofChlamydia major outer membrane protein (MOMP) comprising: (a)transforming a population of E. coli host cells with an expressionvector comprising a nucleic acid molecule comprising a sequence ofnucleotides that encode a leader sequence for targeting the MOMP to theouter membrane of the cell and a sequence of nucleotides that encodeChlamydia MOMP, wherein the nucleic acid molecule is operatively linkedto a promoter; (b) culturing the transformed cells under conditions thatpermit expression of the nucleic acid molecule and translocation to theouter membrane of the cells to produce a recombinant Chlamydia MOMP; and(c) optionally purifying the MOMP.
 2. The method of claim 1, wherein thesequence of nucleotides that encodes the MOMP is codon harmonized orcodon optimized for optimal expression in an E. coli host cell.
 3. Themethod of claim 1 or 2, wherein the leader sequence is operativelylinked to the N-terminus of the MOMP.
 4. The method of any of claims1-3, wherein the leader sequence is directly adjacent to the MOMPsequence.
 5. The method of any of claims 1-4, wherein the nucleic acidmolecule further comprises a sequence of nucleotides that encodes apolypeptide attached to the MOMP, wherein the polypeptide is selectedfrom the group consisting of: a linker, an additional antigen, apolypeptide having adjuvant properties, a polypeptide for facilitatingpurification, a polypeptide for enhancing stability of the MOMP, acarrier protein, and a marker protein.
 6. The method of any of claims1-5, wherein the MOMP comprises a sequence of amino acids as set forthin any of SEQ ID NO:23 through SEQ ID NO:31.
 7. The method of any ofclaims 1-5, wherein the MOMP comprises a sequence of amino acids thatshares at least 90% sequence identity with the MOMP amino acid sequencesset forth in any of SEQ ID NO:23 through SEQ ID NO:31.
 8. The method ofany of claims 1-7, wherein the leader sequence comprises the Shigellaflexneri SopA sequence set forth in SEQ ID NO:8, the Salmonella entericaPgtE sequence set forth in SEQ ID NO:9, the Yersinia pestis Pla sequenceset forth in SEQ ID NO:10, the E. coli OmpP sequence set forth in SEQ IDNO:11, the E. coli OmpA sequence set forth in SEQ ID NO:12, or thepectate lysase B (PelB) sequence set forth in or SEQ ID NO:13.
 9. Themethod of any of claims 1-7, wherein the leader sequence comprises asequence of amino acids that shares at least 90% sequence identity withthe amino acid sequence set forth in any of SEQ ID NO:11, SEQ ID NO:12,or SEQ ID NO:13.
 10. The method of any of claims 1-4, wherein thenucleic acid molecule comprises a sequence of nucleic acids as set forthin SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:20,or SEQ ID NO:21.
 11. The method of claim 1, wherein the nucleic acidmolecule shares 90% or more homology with the nucleotide sequence setforth in any of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:18,SEQ ID NO:20, or SEQ ID NO:21.
 12. The method of any of claims 1-11,wherein the promoter is a low or moderate strength promoter.
 13. Themethod of any of claims 1-12, wherein the expression vector is a vectorthat is associated with a low to moderate transcription or translationrate.
 14. The method of any of claims 1-13, wherein the method furthercomprises a step of inducing the transformed host cell with IPTG forfrom about 4 hours to about 6 hours.
 15. The method of claim 14, whereinthe induction step is carried out at about 30° C.
 16. The method ofclaim 14 or 15, wherein the cell density (OD590) is allowed to reachabout 0.4 to about 0.8 before the induction step is carried out.
 17. Arecombinant MOMP produced by the method of any of claims 1-16.
 18. Apharmaceutical composition comprising an immunologically effectiveamount of the rMOMP of claim 17 and a pharmaceutically acceptablecarrier.
 19. The pharmaceutical composition of claim 18, furthercomprising one or more additional rMOMP antigens.
 20. The pharmaceuticalcomposition of claim 18 or 19, further comprising an adjuvant.
 21. Amethod of inducing an immune response against Chlamydia MOMP in apatient in need thereof, comprising administering a pharmaceuticalcomposition of any of claims 18-20 to the patient.
 22. A nucleic acidmolecule comprising a sequence of nucleotides that encodes Chlamydiamajor outer membrane protein (MOMP) comprising a nucleotide sequencethat is codon harmonized relative to the wild-type nucleotide sequenceencoding the same MOMP.
 23. The nucleic acid molecule of claim 22,further comprising a nucleotide sequence that encodes a leader sequencefor targeting the MOMP to the outer membrane of a cell, wherein theleader sequence is operatively linked to the N-terminus of the MOMP. 24.The nucleic acid molecule of claim 23 which comprises a sequence ofnucleotides as set forth in SEQ ID NO:15, SEQ ID NO:18, or SEQ ID NO:21.25. Use of (a) the rMOMP of claim 17 in the manufacture of a medicamentfor the treatment or prophylaxis of disease associated with Chlamydiatrachomatis infection.
 26. Use of a pharmaceutical composition of anyone of claims 18-20 for the treatment or prophylaxis of diseaseassociated with Chlamydia trachomatis infection.